Mycobacterium tuberculosis (Mtb) infects over one-third of the human population, causing 8 million new cases of pulmonary tuberculosis (TB) each year and ~2.5 million deaths worldwide. The global burden of tuberculosis has been compounded by its deadly association with HIV and by the emergence of multi-drug resistant strains, leading the World Health Organization to declare TB as a global health emergency. The design of new drugs, vaccines and better therapeutics requires identification of the molecular mechanisms governing immune responses and their regulation upon mycobacterial infection. Toll-like receptors (TLR) 2 and TLR4 are mycobacterial important sensors that play a key role in activating innate host responses, as evidenced by an increased susceptibility to mycobacterial infections and impaired cytokine production in mice deficient for these receptors. In humans, the TLR2 (e.g., R753Q) and TLR4 (e.g., D299G) single nucleotide polymorphisms (SNPs) have been correlated with incidence and/or severity of Mtb infections and TB. However, the molecular mechanisms by which such SNPs affect receptor signaling are unknown. Based on our preliminary data, we formulated the hypothesis that the polymorphisms affect recognition of mycobacteria, TLR signalosome assembly and activation of kinases and transcription factors, altering expression of inflammatory mediators, phagocytosis and predisposing to infection in vivo. This hypothesis will be tested in the following Specific Aims to: 1. Define the impact of the TLR2/4 SNPs on TLR expression, localization, signalosome assembly and activation of key kinases and transcription factors in response to mycobacteria; 2. Determine the effect of the TLR2/4 SNPs on expression of inflammatory mediators, mycobacterial phagocytosis and intracellular survival; and 3. Identify the consequences of TLR2/4 SNPs on susceptibility to mycobacterial infections in vivo in transgenic mice expressing orthologs of human mutations. Our studies are expected to identify mechanisms by which the SNPs affect mycobacterial recognition, TLR2/4 signalosome assembly, activation of kinases and transcription factors, and to link these changes to downstream responses important for host defense against mycobacteria, such as expression of inflammatory cytokines and phagocytosis. We will engineer new transgenic mice expressing orthologs of human R753Q TLR2 or D299G TLR4 SNPs to identify the consequences of these mutations on susceptibility and severity of mycobacterial infections. Elucidation of the molecular basis by which TLR2/4 SNPs compromise host defense against mycobacteria and identification of intermediates with compromised functions will pave the way for the development of new approaches and drugs to improve TLR-mediated Mtb sensing and host defense. These advances would be of key importance for improving public health in tuberculosis patients in the U.S.